Methods for producing target cell reactive lymphocytes

ABSTRACT

Methods and compositions for producing target cell reactive lymphocytes, e.g., cytolytic T-lymphocytes, in a subject are provided. In practicing the subject methods, a lymphocyte population is contacted with an effective amount of a target cell peptide mixture of active and inactive peptides to produce lymphocytes reactive, e.g., cytolytic, for the target cell. Also provided are compositions, kits, and systems for practicing the subject methods. The subject invention finds use in a variety of different applications, including therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.60/591,889 filed Jul. 27, 2004; the disclosure of which is hereinincorporated by reference.

GOVERNMENT RIGHTS

This invention was made with government support under federal grant no.NIH R01 CA 090809 awarded by the NIH. The United States Government mayhave certain rights in this invention.

INTRODUCTION

1. Background of the Invention

Interest in the development of peptide-vaccination strategies for cancerhave driven driven by the possibility of inducing tumor associatedantigen (TAA) specific T cell responses that can efficiently eliminate apatients' cancer cells. The anti-tumor potential of TAA-specific CD8⁺ Tcells has been illustrated by the demonstrated capacity of adoptive Tcell therapy to reduce tumor size. While endogenous anti-tumor CD8⁺ Tcell responses may already exist in some cancer patients, vaccinationwith TAA-derived peptides, and in particular heteroclitic peptideanalogs, increases the frequency of TAA-specific T cells to detectablelevels in many patients. However, the presence of TAA-specific T cellselicited by vaccination often does not correlate with clinicalresponses.

There are a large number of strategies to increase the magnitude of Tcell responses to peptide vaccines. These include various adjuvants suchas IFA, IL-12, GM-CSF, anti-CTLA-4 antibodies, and heat shock proteins.Thus far, none of these approaches have improved clinical responses.

As such, there is a continued need for the development of improvedvaccination strategies.

2. Relevant Literature

Bullock et al., J. Immunol. (2003) 170: 1822-1829; Oh et al., J.Immunol. (2003) 170: 2523-2530 and Xu et al., (2003) J. Immunol. 171:2251-2261.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the induction of target cellreactive T-lymphocytes, including cytolytic T-lymphocytes. In practicingthe subject methods, a lymphocyte population is contacted with aneffective amount of a mixture of peptide antigens associated with thetarget cell of interest, where the mixture comprises peptides that areboth active and inactive in the generation of reactive lymphocytes. Alsoprovided are compositions, kits, and systems for practicing the subjectmethods. The subject invention finds use in a variety of differentapplications, including therapeutic applications.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Melanoma patient samples selected for analysis of RE formelanoma cells. a. Six patients with T cell responses specific forMART-1 or gp100 were selected for analysis. PBMC from each patient werestained with PE-conjugated peptide-MHC tetramers: G209-2M-tet PE orM26-tet PE, and co-stained with anti-CD8 FITC and anti-CD14, -CD19, and-CD4 Cy5PE. The plots shown are gated for CD14-, CD19-, and CD4-cells.Tetramer+/CD8+ cells are boxed. FIG. 1B, the estimated percentage oftotal CD8+ cells that are also tetramer+/CD8+ for each patient are: 422:2.5%; 476: 0.31%; 132: 0.22%; 517: 0.23%; 520: 0.12%, 461: 0.50%. b.Micro-cytotoxicity ⁵¹Cr-release assay with tetramer+/CD8+ cells isolatedby FACS from the PBMC from patient 422. Isolated cells were assayed forlysis of T2 cells treated with relevant (G209-2M, G209n) or irrelevant(CMV) peptide, or mel 526 melanoma cells. Sorted cells were combinedwith 250 target cells at 13:1 E:T ratios for 4 hours and supernatantswere assayed for percent specific release of radio-label.

FIGS. 2A-2C. Endogenous cytolytic T cells are more efficient thanvaccine-elicited cytolytic T cells in lysing melanoma cells. Cells from84 clonal CTL lines were assayed for lysis of melanoma cells mel 526,Malme-3M and A-375 in ⁵¹Cr-release cytotoxicity assays. Mel 526 andMalme-3M are HLA-A2.1+ and express both gp100 and MART-1. A-375 cellsare HLA-A2.1+ but do not express either gp100 or MART-1 and served as anegative control. T2 cells treated with 1 μg/ml G209-2M or M26 peptidesserved as controls for antigen-specific lysis. The CTL clones assayedwere selected to represent tetramer+ subsets expressing different T cellreceptor V-beta subunits. Dominating tetramer+ populations in eachpatient were represented with two or more clones. Each CTL clone wasassayed in triplicate wells and the data displayed are averages of twoindependent experiments. CTL clones from the same patient expressingsimilar V-beta subunits which exhibited different lysis potential wereviewed as separate subsets. Each assay was performed at 10:1 effector totarget ratio as detailed in the methods section. FIGS. 2 a-2 b.Efficiency in melanoma cell lysis as a function of relative populationsize. The height of each bar represents % specific lysis, while thewidth represents the relative size of the tetramer+ subpopulations(defined by V-beta expression) in each patient. Population size wasdefined as the percent of CTL clones from each patient expressing thesame V-beta. Error bars show standard deviation (SD) between twoexperiments with each clone and/or between different clones where morethan one clone was analyzed. FIG. 2C. CTL clones derived from eachpatient were classified as “efficient” (>40%), “intermediate” (>10%,<40%) or “poor/no lysis” (<10%) in lysis of melanoma cells. Each barrepresents the portion of total clones from each patient with“efficient”, “intermediate” or “poor/no” melanoma lysis potential.

FIGS. 3A-3J. Endogenous CTL clones have higher RE than vaccine-elicitedCTL clones. CTL clones representing different tetramer⁺ populations ineach patient expressing different V-beta, were assayed for lysis of T2cells pulsed with various dilutions of G209n, G209-2M, M27, or M26peptides in ⁵¹Cr-release cytotoxicity assays. All assays were performedin triplicate and each clone was assayed twice. Error bars reflectvariation between two separate assays. 3a.-c. CTL clones 476.105 and132.1 were assayed for lysis of T2 cells pulsed with 10-fold dilutionsof a. native or c. heteroclitc peptide at concentrations ranging from100 fg/ml to 100 ng/ml. b. CTL clones 476.105 and 132.1 were assayed forlysis of Malme-3M melanoma cells. d.-e. RE scores, equal to the negativelog₁₀ of the peptide concentration that resulted in 40% lysis ofpeptide-pulsed T2 cells, for both (d.) MART-specific and (e.)gp100-specific clones from all patients were correlated with efficiencyin lysing melanoma cells. Correlation coefficients were 0.66 forMART-specific clones and 0.81 for gp100-specific clones. f.-i.Endogenous (461 and 132) and vaccine-induced (517, 520, 422 and 476) CTLclones were compared for RE in lysing target cells. f-g. Target cellswere pulsed with native peptides (f.) M27 and (g.) G209n. Mean RE(weighted) for each response is indicated with horizontal bars. Weightedmeans were based on all clones, not only those assayed, and wereestimated by summing the RE of each analyzed clone multiplied by thenumber of total clones expressing the same V-beta in each patient.Weighted means were: 517: 5.7; 520: 7.0; 461: 7.9; 422: 9.7; 476: 9.9;and 132: 11.2. One-tailed T-tests demonstrated that endogenous CTL hadsignificantly higher RE than vaccine induced CTL: 461 vs 517:p=1.8×10⁻⁵; 461 vs 520: p=1.1×10⁻³; 132 vs 422: p=6×10⁻⁶; and 132 vs476: p=4.3×10⁻⁴. h.-i. Target cells were pulsed with heterocliticpeptides (h.) M26 and (i.) G209-2M. j. CTL clones that were efficient(132.2 and 476.104) or inefficient (422.5F9 and 476.108) in melanomalysis were compared for lysis of target cells pulsed with increasingconcentrations of native or heteroclitic peptides (as in a. and c.).

FIGS. 4A-4B. Target cells pulsed with a combination of agonist and“null” peptides are selectively lysed by high RE but not low RE T cells.High (132.2 and 476.139) and low (422.5F9 and 476.105) RE CTL cloneswere assayed for lysis of T2 cells pulsed with a. 10 ng/ml native G209npeptide (ITDQVPSFV (SEQ ID NO:1); agonist) and various concentrations ofG209-3A peptide (ITAQVPSFV (SEQ ID NO:2); null) at ratios ofagonist:null peptide ranging from 1:1 to 1:10,000, or b. with a constantratio agonist:null peptides of 1:10,000 at total peptide concentrationsranging from 100 ng/ml to 100 μg/ml. Lysis of T2 cells pulsed withagonist only is shown for comparison.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for producing target cell reactive lymphocytes,e.g., cytolytic T-lymphocytes, in a subject are provided. In practicingthe subject methods, a lymphocyte population is contacted with aneffective amount of a target cell peptide mixture of active and inactivepeptides to produce lymphocytes reactive for the target cell. Alsoprovided are compositions, kits, and systems for practicing the subjectmethods. The subject invention finds use in a variety of differentapplications, including therapeutic applications.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

In further describing the subject invention, the methods will bedescribed first, followed by a review of representative applications inwhich the methods find use, as well as a review of representative kitsand systems thereof that find use in practicing the subject methods.

Methods

As summarized above, the subject invention provides methods of producingtarget specific reactive lymphocytes, including target specificcytolytic T-cells, from an initial population of lymphocytes, e.g., anaïve or unstimulated T-cell population. In other words, the inventionprovides methods of making lymphocytes reactive for a specific targetfrom a parent population of lymphocytic cells. By reactive for aspecific target is meant that the cell is cytolytic for the target, ormediates destruction of the target cell by some means, e.g., bysecreting cytokines to activate other T cells, or causing target cellsto undergo apoptosis (suicide) via other cytokines or mediators (e.g.,Fas). By “cytolytic lymphocyte” is meant a non-B lymphocyte thatexhibits cytolytic activity, where cytolytic lymphocytes include, butare not limited to: cytolytic T-cells, natural killer (NK) cells, NKTcells and CD4⁺ T cells, which cells may degranulate and kill targetcells. While in the broadest sense the invention is directed to theproduction of reactive lymphocytes as defined above, in many embodimentsthe methods and compositions of the invention are employed for theproduction of cytolytic T-cells, usually CD8+ T cells. Accordingly, forease of further description of the invention, the invention will now befurther described in terms of methods and compositions for use in theproduction of cytolytic T-cells. However, the invention is not limitedto the production of cytolytic T-cells, but includes the production ofnon-T-cell reactive lymphocytes, and helper T cells, as described above.

By “cytolytic T-cell” is meant a cell that is cytotoxic for a targetcell, i.e., a cell that is specifically reactive with, and capable ofkilling, a target cell, where the target cell may be any undesired cell,e.g. a neoplastic cell; an autoreactive cell such a memory B cell,autoreactive T cell, etc.; a cell chronically infected with a virus orother intracellular pathogen; and the like.

A feature of the cytolytic cells produced by the subject methods incertain embodiments is that the cells have a high Recognition Efficiency(RE) for the target cells for which they are cytolytic. By highRecognition Efficiency is meant that the recognition efficiency or RE ofthe cytolytic lymphocytic cells produced by the subject methods, asdetermined by the protocol described in the experimental section, infra,is at least about 10⁻⁹ to 10⁻¹² M. A further feature of certainembodiments of the subject methods is that when a plurality orpopulation of cytolytic lymphocytes are generated from a parentpopulation of lymphocytes, the generated population of cytolyticlymphocytes is more homogenous with respect to the RE values of itsmember cells as compared to a population of lymphocytes generated usinga homogenous peptide activator agent. In certain embodiments, thestandard deviation with respect to the RE values of the members of thepopulation produced by methods of the subject invention does not exceedabout one log from the RE range of representative embodiments thatprovides for efficient target reactivity (e.g., at least about 10⁻⁹ to10⁻¹² M).

In practicing the subject methods, a parent population of lymphocytes,e.g., T-cells, is contacted with a target cell specific peptide mixtureto produce the desired population of target cell cytolytic lymphocytes.The initial or parent T-cell population may be a naive population, or apopulation that has been pre-treated with another activator, e.g., aheteroclitic peptide composition, so as to be a mixed RE population ofboth high and low RE cells. As described in greater detail below, thecontacting may occur in vitro or in vivo, depending on the particularapplication in which the methods are employed.

By target cell specific peptide mixture is meant a mixture orcombination of two or more different peptides, where the mixtureincludes at least a first peptide that is an active peptide, e.g., anagonist, and at least a second peptide that is inactive, e.g., a null orantagonist peptide. For purposes of the present invention any two givenpeptides are considered to be different or distinct if their sequencesdiffer from each other, when aligned for maximum agreement, by at leastone residue or amino acid. While a given target cell specific peptidemixture may include only two different or distinct peptides, i.e., theactive and inactive peptides as described above, in certain embodimentsthe target cell specific peptide mixture may include three or moredifferent peptides, where the total number of different or distinctpeptides in a given mixture may be as a great as about 10 or greater,e.g., as great as about 25 or greater, where the total number ofdifferent peptides in a given mixture may not, in certain embodiments,exceed about 50.

As indicated above, the peptide mixtures employed in the subject methodsinclude both active and inactive peptides. In many embodiments, activepeptides employed in the subject methods are peptides that are derivedfrom proteins that distinguish or differentiate the target cell fromother cells that can be present in the environment or vicinity of thetarget cell when it is to be contacted with the cytolytic lymphocyteproduced by the present methods. For example, where the target cell is atumor cell, the active peptides of the peptide mixture may be peptidesderived from a tumor associated antigen (TAA) of the target tumor cell.Where the target cell is an autoreactive lymphocyte, the peptides mayinclude antibodies present on the surface of B cells, T cell antigenreceptors present on the surface of T cells; and the like. Where thetarget cell is a chronically infected cell, the peptides may be pathogenproteins, e.g. viral antigens, etc. Specific representative examples ofTAAs are provided in the Experimental Section below.

As is known in the art, active peptides are agonist peptides that, whenpresented by a target cell to a cytolytic T-cell specific for theprotein from which the peptide is derived, cause the T-cell to bind andlyse (react to) the target cell. Active peptides may range in size, andin many representative embodiments range from about 8 to about 16residues in length, such as from about 8 to about 16 residues in length,including from about 6 to about 40 residues in length. Active peptidesmay include immunodominant epitopes of the protein. The active peptidesmay include a sequence that is found in the sequence of the targetprotein, e.g., TAA, or a sequence that is substantially identical to asequence found in the sequence of target protein. By substantiallyidentical is meant a sequence that has at least about 85%, such as atleast about 90%, including at least about 95% sequence identity with asequence of the target protein, where sequence identity is measured bythe BLAST compare two sequences program available on the NCBI websiteusing default settings, as measured over the entire length of theprotein, where the website has the address made up by placing “www.” infront of and “.gov” in back of “ncbi.nlm.nih”.

A given peptide mixture employed in a method according to the subjectinvention may include a single active peptide, or two or more differentactive peptides, where the number of different active peptides in agiven mixture may be as high as 10 or higher, but in representativeembodiments does not exceed about 50.

As indicated above, the peptide mixtures of the present invention alsoinclude inactive peptides. The term inactive peptide is used to describepeptides that are either null peptides or antagonist peptides. As isknown in the art, null peptides are peptides that, when presented by atarget cell to a cytolytic T-cell specific for the correspondingantigen, do not elicit a cytolytic response with respect to the targetcell, such that the cytolytic T-cell does not kill the target cell. Alsoas is known in the art, antagonist peptides are peptides that inhibit acytolytic T-cell from lysing a target cell.

Inactive peptides may range in size, and in many representativeembodiments range from about 8 to about 16 residues in length, such asfrom about 8 to about 16 residues in length, including from about 6 toabout 40 residues in length. The inactive peptides may or may notinclude a sequence that is found in the sequence of the target protein,e.g., TAA, or a sequence that is substantially identical to a sequencefound in the sequence of target protein. By substantially identical ismeant a sequence that has at least about 85%, such as at least about90%, including at least about 95% sequence identity with a sequence ofthe target protein, where sequence identity is measured as describedabove. As such, in certain embodiments the inactive peptides may havesequences that are completely different from any sequence found in thetarget antigen, while in other embodiments the inactive peptides mayinclude a sequence that is the same as or substantially identical to asequence found in the target antigen, e.g., the tumor associate antigenof the target cell. Furthermore, in certain embodiments the inactivepeptides may have sequence similarity to the active peptides, asdescribed above.

A given peptide mixture employed in a method according to the subjectinvention may include a single inactive peptide, or two or moredifferent inactive peptides, where the number of different inactivepeptides in a given mixture may be as high as 10 or higher, but inrepresentative embodiments does not exceed about 50.

The ratio of active to inactive peptides in a given target cell specificmixture employed in the subject methods is chosen to provide for thedesired production of high RE cytolytic T-cells. In representativeembodiments, the ratio of active to inactive peptides in a given mixtureranging from about 1:100 to about 1:100,000, such as from about 1:100 toabout 1:100,000, including from about 1:1 to about 1:10,000,000 (or theinverse ratio).

As indicated above, in practicing the subject methods an initial T-cellpopulation is contacted with a target cell specific peptide mixture in amanner sufficient to produce a desired cytolytic T-cell populationhaving a high RE for the target cell. The conditions under which contactoccurs may be in vitro or in vivo.

For in vitro applications, a T-cell population and target cell specificpeptide mixture are combined and incubated, e.g., in an aqueous mediathat includes one or more needed or desired factors, and maintainedunder conditions suitable for desired production of the product T-cellpopulation to occur. Representative mediums that may be employed includebut are not limited to: currently employed culture mediums, whichmediums may be liquid or semi-solid, e.g., containing agar,methylcellulose, etc. The cell population may be conveniently suspendedin an appropriate nutrient medium, such as Iscove's modified DMEM orRPMI-1640, normally supplemented with fetal calf serum (about 5-10%),L-glutamine, a thiol, particularly 2-mercaptoethanol, and antibiotics,e.g. penicillin and streptomycin. The amounts of reagents employedduring this step may vary and are readily determined by those of skillin the art, where representative parameters are provided in theExperimental Section, below. Following contact, the product compositionis maintained, typically at a temperature ranging from about 36° C. toabout 38° C., including from about 25° C. to about 42° C., for a periodof time ranging from about 1 hour to about 8 hours, including from about0.5 hour to about 24 hours.

For in vivo applications, an effective amount of a peptide mixture isadministered to a subject, e.g., patient, to produce the desired targetcell specific cytolytic T-cells in the subject. The peptide mixture maybe administered by any suitable means, including but not limited to:parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal. Representative Parenteral infusions include, but are notlimited to: intramuscular, intravenous, intraarterial, intraperitoneal,or subcutaneous administrations. In addition, the peptide compositionmay be suitably administered by pulse infusion, including with decliningdoses of the peptide composition. As desired, dosing may be given byinjections, such as intravenous or subcutaneous injections, depending inpart on whether the administration is for a short or long period oftime.

For the prevention or treatment of disease, the appropriate dosage ofpeptide composition will depend on the type of disease to be treated,the severity and course of the disease, whether the peptide compositionis administered for preventive or therapeutic purposes, previoustherapy, a given patient's clinical history and response to the peptidecomposition, and the discretion of the attending health careprofessional. The peptide composition is suitably administered to thepatient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 mg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of peptide composition is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 mg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment may be sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

The peptide composition may be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the peptide composition to beadministered will be governed by such considerations, and is the minimumamount necessary to prevent, ameliorate, or treat a disease or disorder.The peptide composition need not be, but may optionally be formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of peptide mixture present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

Practice of the subject methods results in the production of cytolyticT-cells as described above. Specific representive cytolytic T-cells ofinterest that may be prepared by the subject methods include, but arenot limited to: T-cells that are cytolytic, i.e., capable of killing orcytotoxic for, a wide variety of different types of target cells, suchas disease causing cells, e.g., hazardous/pathogenic cellularmicroorganisms, such as Pneumococcus, Staphylococcus, Bacillus,Streptococcus, Meningococcus, Gonococcus, Eschericia, Klebsiella,Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Yersinia,Listeria, Corynebacterium, Vibrio, Clostridia, Chlamydia, Mycobacterium,Helicobacter and Treponema; protozoan pathogens, and the like; whereintracellular pathogens are of particular interest, as well as diseasecausing cells endogenous to the host, e.g., autoreactive cells,neoplastic cells, including cancerous cells, and the like. Specificrepresentative neoplastic target cells include those found in thefollowing representative types of cancers: melanomas, carcinomas, suchas squamous cell carcinomas, adenocarcinomas, transitional cellcarcinomas, basal cell carcinomas, etc., which may include colon,duodenal, prostate, breast, melanoma, ductal, hepatic, pancreatic,renal, endometrial, stomach, dysplastic oral mucosa, polyposis, invasiveoral cancer, non-small cell lung carcinoma, transitional and squamouscell urinary carcinoma, lymphomas and leukemias, gliomas, astrocytomas,sarcomas, etc.

The subject methods find use in a variety of different applications,where representative applications are reviewed in greater detail below.

Utility

The subject methods find use in a variety of different applicationswhere one wishes to produce reactive lymphocytes, e.g., cytolyticT-cells. One representative application in which the subject methodsfind use is in therapeutic protocols to produce therapeutic agents,e.g., therapeutic cytolytic T-cells. In such applications, the methodsare employed to produce a cytolytic T-cell population that is specificfor a target cell type that is responsible for a disease conditionafflicting a subject or patient. As convenient, the cytolytic T-cellpopulation may be produced in vitro and then administered to a host, orproduced directly in the host, such that the peptide mixture isadministered to the host as a vaccine.

In those in vitro embodiments, the product population may be furtherprocessed or screened to identify and isolate those cells of the productpopulation that have desirable cytolytic properties. For example, themethods described in U.S. application Ser. No. 60/530,798 (thedisclosure of which is herein incorporated by reference) may be employedto screen and isolate cytolytic cells from the product population of invitro embodiments of the subject methods. Briefly, in the isolationmethods described in the Ser. No. 60/530,798 application, a sample iscontacted with a target cell stimulator, e.g., a neoplastic cell, and adetectably labeled granule membrane protein specific binding agent.Following contact, any resultant labeled lymphocytes, e.g., T-cells, areidentified as lymphocytes cytolytic for the target cell. The resultantenriched isolated T-cell composition may then be expanded ex vivo toproduce an increased population of cytolytic T-cells. In certainembodiments, a feature of the subject methods is that the harvestedpopulation of cells is expanded, where the expansion step occurs at somepoint in time prior to reintroduction of the cells to the subject oforigin. In the expansion step, the number of T-cells in the harvestedcell collection is increased, e.g., by at least about 4 fold, such as byat least about 4 fold as compared to the originally isolated amount,such that at least in certain embodiments the final number may be fromabout 100- to about 100,000-fold or more greater than the originalnumber of cells. As such, the isolated cells are proliferated to producean expanded population of harvested T-cells.

The isolated cells may be proliferated in this step according to anyconvenient protocol. For example, the cells are proliferated or enhancedby contacting the cells with an expansion agent, by which is meant anagent that increases the number of cells by causing cellularproliferation. A variety of different such agents are known, whererepresentative agents include, but are not limited to: growth factors,accessory cells, ligands of specific activation receptors that may bemonoclonal antibodies or antigens, and the like. One representative suchprotocol is described in U.S. Pat. No. 6,352,694; the disclosure ofwhich is herein incorporated by reference.

Depending on the particular embodiment being practiced, an effectiveamount of the peptide mixture or in vitro cytolytic T-cells producedthereby is administered to the host. By effective amount is meant anamount effective to achieve the desired treatment of the host. Bytreatment is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g. prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.

In certain embodiments, the subject/host/patient being treated may havebeen pretreated with an initial vaccine, e.g., comprising heterocliticpeptides, in order to initially stimulate or activate a naive cytolyticT-cell population.

A variety of hosts are treatable according to the subject methods. Incertain embodiments, such hosts are “mammals” or “mammalian,” wherethese terms are used broadly to describe organisms which are within theclass mammalia, including the orders carnivore (e.g., dogs and cats),rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g.,humans, chimpanzees, and monkeys). In many embodiments, the hosts willbe humans.

Pharmaceutical Formulations

Therapeutic formulations of the peptide mixtures of the subjectinvention are also provided. As desired, the peptide mixtures may beprepared for storage by mixing the peptide mixtures having the desireddegree of purity with optional physiologically acceptable-carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of lyophilized formulationsor aqueous solutions. Acceptable carriers, excipients, or stabilizersare nontoxic to recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, such as thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration are generallysterile, where sterility may readily be accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide variant, which matricesare in the form of shaped articles, e.g., films, or microcapsule.Examples of sustained-release matrices include polyesters, hydrogels(for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Kits & Systems

Also provided are kits and systems that find use in practicing thesubject methods, as described above. For example, kits and systems forpracticing the subject methods may include one or more pharmaceuticalformulations, which include one or both of the peptide components of theabove-described peptide compositions or mixture, i.e., the inactive andactive peptide components. As such, in certain embodiments the kits mayinclude a single pharmaceutical composition, present as one or more unitdosages, where the composition includes both the active and inactivepeptides of the above described mixtures. In yet other embodiments, thekits may include two or more separate pharmaceutical compositions, eachcontaining either an active or inactive peptide component.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The term “system” as employed herein refers to a collection of activeand inactive peptide components, present in a single or disparatecomposition, that are brought together for the purpose of practicing thesubject methods. For example, separately obtained active and inactivepeptides brought together and coadministered to a subject, according tothe present invention, are a system according to the present invention.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

I. Methods

A. Patient samples. Peripheral blood mononuclear cell (PBMC) sampleswere isolated from melanoma patients after vaccination with theheteroclitic peptides MART 26-35 (27L) (ELAGIGILTV (SEQ ID NO:3)) andgp100 209-217 (210M) (IMDQVPSFV (SEQ ID NO:4)) in incomplete Freund'sadjuvant (IFA) at the USC Norris Cancer Center, Los Angeles, Calif.under an IRB approved protocol. PBMC samples were stored at −130° C. andwere thawed the day before an experiment for overnight culture in CTLmedia. The following morning, viable cells were isolated by ficolldensity centrifugation, washed, and resuspended to the appropriateconcentration in 90% Iscove's Modified Dulbecco's medium (IMDM), 10%fetal bovine serum (FBS).

B. Flow Cytometry Analysis. For detection/isolation of peptide-specificT cells, patient PBMC samples were stained and analyzed by FACS aspreviously described Lee et al., Nat. Med. (1999) 5: 677-685. Briefly,cells were stained with anti-human CD8− fluorescein isothiocyanate(FITC) (Caltag) and CD19-CyChrome (BD Biosciences) antibodies (Ab), andHLA-A*0201/peptide tetramer-phycoerythrin (PE). The final stainingdilution of each Ab was 1/200 and 1/80, respectively. Tetramer-PE wastitrated for optimal staining, usually between 1 and 10 μg/ml. For TCRVbeta-typing, cells were divided in 7 aliquots and stained with CD8PerCP-Cy5.5 (BD Biosciences), tetramer-PE, and a panel of 2 or 3different anti-Vbeta mAb labeled with FITC, allophycocyanin (APC), orboth. Cells were incubated at room-temperature for 30 mins, washed, thenanalyzed using a two-laser, 4-color FACSCalibur (Becton Dickinson, SanJose, Calif.), or sorted using a FACSVantage flow cytometer (BectonDickinson, San Jose, Calif.). Lymphocytes were identified by forward andside scatter signals, then selected for CD8+ and tetramer+. Up to onemillion events were acquired and analyzed using FlowJo (TreeStar, SanCarlos, Calif.).

C. Generation of CTL clones. CD8+ T cell clones were obtained byFACSorting individual tetramer+ cells from PBMC samples prepared forflow cytometry as described above. CD8+ tetramer+ T cells were sorted atone cell per well into 96 well plates containing 100 μl of CTL media(IMDM, with 10% FBS, 2% human AB sera, and Penicillin, Streptomycin, andL-Glutamine, supplemented with 100 U/ml IL-2) under sterile conditionsusing a FACS Vantage (Becton Dickinson, San Jose, Calif.). Sorted cellswere expanded in vitro using standard protocols. Briefly, irradiatedfeeder cells (JY cells and fresh PBMCs) were added to wells containingthe sorted T cells and the 96 well plates were incubated at 37° C., 7%CO₂ to allow for growth. Potential clones become visible around day 14and were then transferred to 24 well plates containing 1 ml CTL mediawith 100 U/ml IL-2. Wells were selected based on cell confluency forexpansion and further analysis. Clones confirmed to be tetramer+ wereexpanded in T-25 flasks containing irradiated JY cells and fresh PBMCsin 25 ml CTL media containing PHA. IL-2 was added to a finalconcentration of 50 U/ml on day 1 and then every 2 days thereafter for 2weeks.

D. Cytotoxic Assays. 1. Target Cells: The HLA-A*0201+ melanoma linesMalme-3M and A375 were purchased from ATCC and maintained according totheir instructions. The HLA-A*0201+ melanoma line mel526 was a kind giftfrom Dr. Y. Kawakami. While Malme-3M and mel526 express both MART andgp100, A375 does not express MART or gp100 and served as a negativecontrol. Expression (or lack of) of these antigens by each cell line wasfurther confirmed by immunohistochemical staining. These cells adhere toplastic and were trypsinized using Trypsin/EDTA solution (Gibco) beforeuse. They were washed and resuspended to the appropriate concentration(usually 10⁵/ml) in 90% IMDM, 10% FBS.

2. Determination of recognition efficiency (RE): ⁵¹Chromium(⁵¹Cr)-labeled T2 targets were pulsed with a range of peptideconcentrations, generally starting at 10⁻⁷ M and decreasing by log stepsto 10⁻¹³ M. T cell clones were incubated with T2 targets at 10:1 E:Tratios for 4 hours, then ⁵¹Cr release was measured and percentagecytotoxicity calculated by standard methods. Prior to each cytotoxicityassay, clones underwent ficoll-hypaque centrifugation to remove deadfeeder cells, and were determined to be >80% CD8+ tetramer+ T cells byFACS. The E:T ratio was based upon live T and target cells. For each Tcell clone, % cytotoxicity was plotted against peptide concentration.The peptide concentration at which the curve crosses 40% cytotoxicitywas defined as the RE of that clone (Marguiles, Nat. Immunol. (2001)2:669-670.

3. Microcytotoxic assay: Cells were isolated directly from PBMC frompatient 422 by FACS as described above. Cells were collected inmicrofuge tubes containing 1 ml ice cold 90% IMDM, with 10% FBS.Collected cells were washed and resuspended to 83,300 cells/ml in 90%IMDM, with 10% FBS. Targets were prepared as described above andresuspended to 8,300 cells/ml in 90% IMDM, with 10% FBS. 2,500 sortedcells (30 μl) and 250 target cells (30 μl) were transferred to amicrocentrifuge tube (VWR), centrifuged 1 min at 200×g and incubated 4hours at 37° C. Percent specific release of ⁵¹Cr was calculated from 40μl cell-free supernatant.

II. Results

A. T cell responses to tumor-associated antigens (TAAs) in melanomapatients. To address the complexity of T cell responses against melanomain vivo, patients with vaccine-induced or endogenous tumorantigen-specific responses were selected. In recent cancer vaccinetrials, many melanoma patients who received heteroclitic peptidevaccines gp100 _(209-217 (210M)) (IMDQVPSFV (SEQ ID NO:4); G209-2M) andMART-1 _(26-35 (27L)) (ELAGIGILTV (SEQ ID NO:3); M26) had measurableCD8+ peptide-specific T cell responses in PBMC detected by peptide-MHC(pMHC) tetramer staining. In addition, TAA-specific T cell responsescould be detected in some patients without vaccination, suggesting theexistence of an endogenous anti-tumor T cell response in these patients.

For the current study, we selected samples from six melanoma patientsfrom these trials—four with vaccine-elicited responses (samples 422,476, 517, and 520) and two with endogenous T cell responses (132 and461)—for detailed analyses of TCR V-beta usage, RE for the targetpeptide, and tumor cytotoxicity. These six patient samples hadpeptide-specific T cell populations detectable with G209-2M-tetramers(patients 422, 476 and 132) or M26-tetramers (patients 517, 520 and 461)ranging from 0.1 to 2.5% of total CD8+ T cells (FIG. 1 a).

B. Vaccine-elicited T cells are cytolytic directly ex vivo. Patient 422had the largest detectable TAA-specific CD8+ T cell response (2.5%G209-2M tetramer+), and thus sufficient numbers for examination of lyticfunction immediately following isolation. To test whether peptidevaccine-induced T cell responses were functionally active directly exvivo, T cells isolated by tetramer-guided cell sorting from patient 422were tested for lysis of peptide-pulsed and melanoma target cells inmicro-cytotoxic assays (FIG. 1 b). The directly isolated tetramer+ Tcells from this patient specifically lysed T2 cells pulsed with highconcentrations (1 μg/ml) of G209-2M- and native (G209n) peptides, butnot with M26-pulsed or melanoma targets. This result shows that while asignificant portion of the vaccine-elicited T cells from patient 422 maybe function in vivo, they did not have significant tumor lysis activity.

C. Vaccine-elicited T cells have varied capacity to lyse melanomatargets. We reasoned that analysis of a set of clonal CTL lines thatrepresented the tetramer+ population would provide an accurate estimateof the complexity of the TAA-specific T cell response in each patient. Alarge number of clonal CTL lines (>200) were generated by fluorescenceactivated cell sorting (FACS) of individual tetramer+ cells directlyfrom PBMC samples (Table 1).

TABLE 1 CTL clones established from each patient represent a randomselection from the tetramer-reactive CD8+ parent population. TCR^(a)Patient 422 Patient 476 Patient 132 Patient 461 Patient 517 Patient520 VB ^(b)CTL ^(c)Tet+ CTL Tet+ CTL Tet+ CTL Tet+ CTL Tet+ CTL Tet+family clones PBMC clones PBMC clones PBMC clones PBMC clones PBMCclones PBMC VB1 3  1  1% 7% 1 8% 1 VB2 1 5% 3% 1 VB3 2 3% 2 2 16%  1 10 VB5.1 1 5% VB7 3% 2 5% VB8 7 5% 1  4% 1 4 4% VB9 1 nt nt nt nt nt nt ntnt nt nt nt VB12 2 2% 5% VB13.1 1 2%  4% 2% 14%  5 11%  VB13.6 1% 7% 7%VB14 6 17%  44 24% 4 13%  1 8% 8 9% VB16 2% 1 1% VB17 7 11%  33 37% 1086% 3% 6% 8 4% VB20 1 1% 5% VB21.3 12 4% 2  2% VB22 3% 1 VB23 1 2 3% VB?17 3 3 3 7 Total: 61 46%  85 72% 11 87% 10 61%  9 66%  48  32% ^(a)Clonal CTL lines were established from each patient ^(b)Number ofclonal CTL lines from each patient expressing the same The same TCR VBchain. ^(c)Percent of tetramer-reactive CD8+ T cells in each patientexpressing the indicated TCR VB chain.

Up to 85% of sorted cells expanded in various sorts. Randomly selectedexpanding clones and the tetramer+ population from which they werederived were examined for TCR V-beta expression using TCRV-beta-specific monoclonal antibodies. Peptide specificity and CD8expression of each clone was confirmed by staining with tetramers andanti-CD8 mAb (data not shown). To obtain an accurate reflection of thetotal T cell population detected with tetramer in each patient, wedecided to rigorously examine at least one representative clone for eachsubpopulation expressing a different TCR V-beta (Table 2).

Multiple clones were analyzed to determine dominating populations.Clones with unknown TCR V-beta expression (not reactive with any of theanti-TCR V-beta mAb) were also included in the analysis (Table 2). Frompatients 132, 517 and 461, for which fewer clones were generated, allclones were included in analysis (Table 2).

To determine the effectiveness of tumor lysis by the differentTAA-specific T cell clones that were propagated, clones were analyzedfor their ability to lyse melanoma cell lines mel 526 and Malme-3M. Bothexpress gp100 and MART-1 melanoma associated antigens and are HLA-A*0201positive while the third line, A-375, does not express either gp100 orMART-1 and served as a control for antigen-specific killing. Inaddition, each CTL clone was examined for antigen specific lysis of T2cells pulsed with high levels (1 μg/ml) of G209-2M or M26 peptides.“Efficient lysis” in these experiments was set at 40% or more specificrelease of radiolabel from the target cells. 10% or less specificrelease was categorized as “poor or no lysis”, and 10% to 40% was termed“intermediate lysis”. All but two of the CTL clones elicited fromendogenous anti-tumor responses (from patients 132 and 461) exhibited“efficient lysis” of both the mel 526 and Malme-3M melanoma cell lines(FIG. 2 a). In contrast, only a few clones from the vaccine-elicitedresponses (from patients 422, 476, 520 and 517) efficiently lysedmelanoma cells. The majority of clones examined from thesevaccine-elicited responses either failed to lyse melanoma targetsaltogether or lysed them with intermediate efficiency (FIG. 2 a). Thislack of efficiency in melanoma cell lysis was not due to cellulardysfunction since each clone efficiently lysed T2 cells pulsed with highlevels of relevant, but not irrelevant, peptide (FIG. 2 a). Overall, themajority of clones derived from endogenous anti-tumor responses(patients 132 and 461) lysed both mel 526 and Malme-3M melanoma targetcells more efficiently compared to clones from vaccine-elicitedresponses (patients 422, 476, 520 and 517), FIG. 2 b. These findingsshow that TAA-specific T cells elicited by heteroclitic peptidevaccination have different tumor-cytolytic potentials from those whichdevelop endogenously.

TABLE 2 CTL clones from each patient selected for functional analysis.Patient 517 Patient 520 Patient 461 Patient 422 ^(a)TCR TCR TCR TCRClone: VB: ^(b)Assay: Clone: VB: Assay: Clone: VB: Assay: Clone: VB:Assay: 517.1 VB7 M 520.17 VB14 MR 461.4 VB2 MR 2A12 VB5.1 MR 517.2 VB?MR 520.21 VB14 MR 461.8 VB3 MR 2C1 VB14 M 517.3 VB1 MR 520.20 VB3 MR461.9 VB14 MR 2E1 VB? MR 517.7 VB7 MR 520.24 VB3 MR 461.10 VB3 MR 3H3.1VB20 MR 517.11 VB14 MR 520.30 VB17 MR 461.17 VB14 MR 4A6 VB9 MR 517.13VB3 MR 520.32 VB17 MR 461.21 VB? MR 4F1 VB1 MR 517.14 VB8 MR 520.22 VB?MR 461.24 VB14 MR 5F9 VB8 MR 517.16 VB? MR 520.31 VB? MR 461.25 VB14 MR1A12 VB21.3 M 517.40 VB? MR 520.33 VB? MR 461.29 VB? MR 422.T1 VB13.1 MR520.38 VB? MR 461.30 VB? MR 422.23 VB14 M 520.41 VB? MR 422.50 VB14 MR520.55 VB? MR 422.47 VB17 MR 520.16 VB8 MR 422.66 VB17 M 520.18 VB16 MR422.27 VB12 MR 520.19 VB13.1 MR 422.49 VB3 MR 520.43 VB2 MR 3F2 VB? MR520.49 VB23 MR 2H9 VB? MR 520.52 VB1 MR 4E2 VB? MR 520.59 VB22 MR 3H3.2VB? MR 422.64 VB8 M 422.72 VB3 M 422.T8 VB1 M Patient 476 Patient 132TCR TCR Clone: VB: Assay: Clone: VB: Assay: 476.101 VB14 MR 132.1 VB17MR 476.102 VB14 M 132.2 VB17 MR 476.105 VB14 MR 132.3 VB17 M 476.108VB14 MR 132.4 VB17 M 476.133 VB14 M 132.5 VB17 M 476.104 VB17 MR 132.6VB1 MR 476.125 VB17 MR 132.9 VB17 M 476.137 VB17 M 132.10 VB17 M 476.139VB17 M 132.11 VB17 M 476.140 VB17 M 476.15 VB21.3 MR 476.110 VB3 MR476.N11 VB3 M 476.25 VB8 MR 476.N8 VB? MR 476.28 VB? MR 476.114 VB? MR476.26 VB21.3 M ^(a)The TCR VB usage of each CTL clone was determinedusing a panel of 19 anti-VB mAbs by flow cytometry. ^(b)All linesselected for functional analysis were assayed for lysis of melanomacells (M). Some lines were also subjected to RE analysis (MR).

D. RE for native and heteroclitic peptides of T cells from endogenous orvaccine-elicited responses. We hypothesized that CTL clones that did notefficiently lyse melanoma targets may be incapable of recognizing therelatively low surface densities of native peptide present on tumorcells. CTL clones selected for analysis of tumor lysis were alsoassessed for RE for the native and heteroclitic peptides via a 10 logrange of dilutions. This is illustrated with clones 132.1 and 476.105(FIG. 3 a). There were considerable differences in killing ofpeptide-pulsed T2 cells by these two clones. The relative differences inRE for G209n native peptide displayed by the two clones highlighted inFIG. 3 a correlated with their ability to lyse melanoma cells: the highRE clone 132.1 efficiently lysed melanoma targets whereas the low REclone 476.105 did not (FIG. 3 b). In contrast to the differences in REfor G209n peptide, similar assays revealed little difference in RE ofthe two clones for G209-2M heteroclitic peptide (FIG. 3 c), showing thatthese clones recognize the native and heteroclitic peptides differently,and that RE for the native, but not heteroclitic, peptide correlateswith tumor-lytic potential.

Similar RE assays were performed for the remaining clones from eachpatient selected for analysis. In order to compare REs of various CTLlines, each clone was assigned an RE score expressed as the negativelog10 value of the peptide concentration required for 40% percentspecific lysis at an E:T ratio of 10:1. For clones 132.1 and 476.105these scores were 11.1 and 8.3 for assays with G209n peptide (FIG. 3 a),and 11.2 and 11.2 for assays with G209-2M heteroclitic peptide (FIG. 3c), respectively. We compiled the data on clones from all patients,which showed a strong correlation between tumor-lytic potential and REfor native peptide (FIGS. 3 d and e). Overall, clones generated fromendogenous anti-tumor responses had much higher RE for the nativepeptide than clones generated from post-vaccine responses (FIGS. 3 f andg). We estimated the composite RE of the overall TAA-specific response(composed of a heterogeneous population of T cells) in vivo by summingthe RE of each clone multiplied by its representation in the originalmixture (the representation was estimated based on the proportion ofTAA-specific cells expressing the same V-beta as the clone). These arerepresented as horizontal bars for each response. Clearly, theendogenous responses (461 and 132) had a higher overall, and morehomogeneous, RE for the native peptide than the vaccine-elicitedresponses (422, 476, 517, 520), FIGS. 3 f and g. Importantly, thevaccine-elicited clones also exhibited wide variations in RE for theheteroclitic peptide as compared to the endogenous clones (FIGS. 3 h andi). These findings show that the variation in RE for native peptides,and hence ability to lyse tumor, for vaccine-elicited CTLs is not merelya reflection of differential recognition of native and heterocliticpeptides by many clones. Rather, variations in RE are a function of themanner in which these cells were elicited by vaccination.

Many clones recognized native and heteroclitic peptides differently,such as 422.5F9 and 476.108 (FIG. 3 j). These clones failed to lysetumor targets (FIG. 2 a). In contrast, some clones, such as 132.2 and476.104, recognized heteroclitic and native peptide with similarefficiency (FIG. 3 j) and efficiently lysed tumor cells (FIG. 2 a). Manyclones were also generated which had low RE for both native andheteroclitic peptides and were inefficient in tumor lysis.

E. Selective stimulation of high RE T cells with peptide mixtures. Todevelop a vaccine strategy which may selectively stimulate T cells ofhigh RE in vivo, we devised a novel strategy in which an agonist peptideis mixed with an analogue peptide with null activity and similar HLAbinding affinity. G209-3A represents such an analog for G209. Thisanalog peptide, which differs from the native peptide by a substitutionof an asparagine for an alanine at residue 211, has similar affinity toHLA-A2.1 as the native peptide but does not activate G209-specifictarget cell lysis by T cell clones at any concentrations (data notshown). When G209n and G209-3A peptides were combined at various ratiosfor pulsing of T2 target cells, we found that a 1:10,000 ratio of nativeto null peptide abolished lysis of T2 cells by low RE CTl clones butlysis by high RE CTL clones was preserved (FIG. 4 a). Importantly, thiseffect was observed with a wide range of peptide concentrations spanningat least two logs (FIG. 4 b), showing that high RE CTL clones may beselectively stimulated using such a strategy across a broad range ofphysiologically achievable peptide concentrations in vivo.

III. Discussion

To achieve maximal clinical responses, the majority of T cells elicitedby vaccination in cancer patients should be capable of lysing tumortargets. We have undertaken the most detailed analysis to-date, on asingle cell level, of cytolytic T cell responses elicited by cancervaccination and compared these with endogenous anti-tumor CTL responses.CTL clones were selected directly from patient PBMC samples withoutenrichment in culture to closely reflect the composition of theantigen-specific T cell response in vivo at the time of isolation. Ourdata revealed that T cell populations induced by vaccination werestrikingly different from endogenous populations: while some CTLelicited by vaccination could kill melanoma targets, most wereinefficient in tumor cell lysis. In contrast, nearly all CTL clones fromendogenous responses were efficient at melanoma cell lysis. Thisdifference was directly related to RE. Clones that did not lyse tumorcells required up to 10³-fold higher concentration of peptide forsimilar levels of lysis of T2 targets compared to T cell clones thatwere tumor-lytic. Side-by-side comparison of endogenous CTL andvaccine-induced CTL suggested that the activation of low RE TAA-specificCTL was selectively driven by heteroclitic peptide vaccination. Thus,high doses of peptide and/or the higher levels of expression ofheteroclitic peptide on APC may induce and actively propagatepredominantly T cells with too low RE for recognition of physiologicallevels of the native peptide present on tumor targets. These data showan inverse relationship between antigen density and the RE of T cellselicited.

Differential recognition of native and heteroclitic peptides by many Tcells may also account for the induction of non-tumor-lytic CTL clonesby heteroclitic peptide vaccines. However, our data suggest that epitopedensity may be the dominant driving factor for RE in vivo. In all of thevaccine-elicited T cell responses, many of the T cells generated wereeither of low or intermediate RE not only for the native peptide, butalso for the heteroclitic peptide, and exhibited no or intermediatelysis of tumor targets. In contrast, nearly all of the clones generatedfrom the endogenous responses were of high RE. This finding shows thatthe high dosage of peptides administered in vaccinations and theincreased binding capacity of heteroclitic peptides to MHC molecules—thevery quality that provides them with increased immunogenicity—drive theinduction of many T cells with low RE for both heteroclitic and nativepeptides.

Another implication of this study is that the number of cells measuredby current methods, including ELISPOT or staining with MHC tetramers,may not correlate directly with the RE or tumor-cytolytic potential of Tcell responses to vaccination. For example, of the nine clones analyzedfrom patient 517, none were efficient in tumor cell lysis, yet thesecells were detectable by MHC tetramer staining. T cells with low RE fornative TAA do not efficiently lyse tumor, and therefore are unlikely tohave an impact on clinical outcome. Furthermore, it may be possible thatlow RE TAA-specific T cells may interfere with elicitation of high RE Tcells either by direct competition for antigen on APC surface ordown-modulation of peptide-MHC complexes.

Our data show that not only quantity, but quality, of the T cellresponse elicited by vaccination is critical to clinical efficacy. Toselectively activate T cells of high RE, we developed a novel approachin which T cells are stimulated with a combination of agonist and nullpeptides. By combining peptides with similar HLA binding properties,there is no selective advantage in APC uptake between peptides, hencethe ratios would be preserved. We showed that at certain ratios, thereis a wide dynamic range of peptide concentrations at which high RE Tcells are selectively activated over low RE T cells. This approachcircumvents the unpredictable nature of peptide trafficking and uptakein APC. Furthermore, this strategy mimics what naturally arises fromtumor cells in vivo: APC that phagocytize apoptotic tumor cells presenta vast mixture of peptides—cognate peptides likely exist with a vastexcess of null peptides. This may be the mechanism by which high RE Tcells are selectively expanded in endogenous responses, and our novelvaccination strategy may replicate this natural mechanism of REselection in vivo.

In certain embodiments, a complete vaccination strategy will involve aninitial induction phase, followed by progressive shaping of the responseto higher RE. Although heteroclitic peptide vaccination may drive Tcells of mixed high and low RE, such a strong stimulus is desirable incertain instances to induce an initial de novo T cell response. Thus,naïve TAA-specific T cells, with inadequate RE to become activated bylow densities of native peptides present on tumor cells, may becomeefficient in tumor lysis upon vaccination with heteroclitic peptide.Therefore, optimized use of heteroclitic peptide to induce an initialpeptide-specific T cell response, followed by selective expansion of thehighest RE tumor-lytic T cells using combinations of native and analogpeptides, according to the present invention, is a particularlyeffective strategy with clear clinical application in certainembodiments.

In summary, we have demonstrated that vaccination with heterocliticpeptide at high concentrations drives T cell responses of predominantlylow RE, and that only high RE T cells are effective at lysing tumor.This may be a key factor in the lack of correlation betweenimmunological and clinical responses after vaccination. Importantly, thesituation is distinctly different in endogenous responses, in which theCTLs generated are predominantly of high RE. This suggests that themanner in which T cells are elicited is different in these two settingsand underlie their differences biologically. The present inventionprovides a novel modification of peptide vaccine strategy that preservesthe initial immunogenic properties of heteroclitic peptide vaccinationwhile selectively expanding high RE T cells. Such a strategysignificantly improves the efficacy of cancer vaccination therapies.

It is apparent from the above results and discussion that the subjectinvention provides convenient protocols for producing high RE cytolyticcells. Accordingly, the subject invention is capable of producing cellsthat are truly cytolytic for a target cell as it naturally occurs, andnot just a cell pulsed with the target peptide. As such, the subjectinvention represents a significant contribution to the art.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of producing tumor cell-reactive T-lymphocytes, said methodcomprising: contacting a lymphocyte population with an effective amountof a peptide mixture comprising a native agonist peptide from atumor-associated antigen, wherein the agonist peptide comprises theamino acid sequence (SEQ ID NO:1) ITDQVPSFV and an analog peptide thatcomprises the sequence (SEQ ID NO:2) ITAQVPSFV; to produce T-lymphocytesreactive for said tumor cell.